Investigations of epithelial cell lineage determination and molecular mechanisms of cell differentiation are crucial for a comprehensive understanding of lung development, injury/repair, and carcinogenesis. Our long-term objective is to clarify these mechanisms, beginning with the concept of the pluripotential stem cell. Epithelium in the lung is composed of pulmonary neuroendocrine cells (PNECs) and non-neuroendocrine cells (NECs), including Clara cells and alveolar type II cells. In rodent, PNEC hyperplasia can be associated with non-NE lung tumors, suggesting that PNECs and non-NECs might have a common precursor cell. Similar to the hematopoietic and nervous systems, specific differentiation of a pluripotential precursor cell could involve active repression of non-specific genes. In Drosophila, neurogenic genes can act as repressors for neural differentiation. Because PNECs play a role in promoting cell growth and differentiation during lung development and injury, and yet remain poorly understood, we are focussing on the role of such neurogenic genes in PNEC differentiation during embryogenesis. We will then analyze how an inflammatory cytokin, tumor necrosis factor-alpha (TNF), can regulate neurogenic genes because TNF is known to induce NE differentiation. We now address three hypotheses with the following Specific Aims: AIM 1: To test the hypothesis that cell lineages of PNECs and non-NECs are derived from a common precursor cell in cultured developing embryonic murine lung buds by microinjection of a retroviral library followed by genetic clonal analysis using PCR. AIM 2: To test the hypothesis that PNEC lineage determination is regulated in part by repression of genes that inhibit neuronal/NE differentiation, such as Notch-1. We will use Notch antisense DNA and retroviral constructs expressing constitutively activated Notch and/or dominant negative Notch to determine whether NE differentiation can be suppressed by Notch activation and driven by Notch repression. AIM 3: To test the hypothesis that cytokines can modulate pulmonary NE cell differentiation by regulating expression of neurogenic genes such as Notch-1 in lung buds. We will focus on TNF which decreases Notch-1 mRNAs coincident with NE differentiation. Cellular processes altered by Notch-1 antisense oligonucleotides will be compared to effects of TNF in vitro. Notch-1 expression and PNEC differentiation will then be assessed in TNF-over-expressing transgenics and TNF-knockout mice. These investigations will open doors to new roads of understanding molecular mechanisms of mammalian lung development.